This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Peptidylglycine ?-hydroxylating monooxygenase (PHM) is a non-coupled binuclear Cu protein, which catalyzes the stereospecific hydroxylation of the ?-carbon of the C-terminal glycine of all peptidylglycine substrates. In the catalytic mechanism, PHM binds and activates O2 for H-atom abstraction from the peptidylglycine substrate. Crystal structure of a pre-catalytic O2 intermediate is available, which demonstrates an end-on bound Cu-O2 species at the active site. We propose to pursue single-crystal Cu K-edge XAS investigations on this pre-catalytic intermediate to determine its geometric and electronic structure. We plan to carry out a time-series photoreduction investigation on the pre-catalytic intermediate to determine its stability upon exposure to the x-ray beam. We also propose to investigate the fully oxidized (CuIICuII) and fully reduced (CuICuI) states of PHM in the crystalline form to characterize their geometric structure and the ligand field strength using a combination of single-crystal XAS and computational methods (density functional theory (DFT) and time-dependent DFT methods). These spectroscopic and computational investigations will enable the electronic structure determination of relevant states in the PHM substrate hydroxylation mechanism. Near-edge multiple scattering analysis will be used to differentiate the two Cu sites present in PHM to determine the binding mode of O2 to Cu in the precatalytic intermediate. These data will also enable the determination of the role of the O2 in the intramolecular electron transfer from the CuH site to the CuM site. The combined analysis of Cu K-edge data, EXAFS, Near-edge multiple scattering analysis, DFT and time-dependent DFT calculations will lead to a better understanding of O2 binding, activation and substrate hydroxylation by PHM.